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Enhanced Performance of an Affinity Biosensor Interface Based on Mixed Self-Assembled Monolayers of Thiols on Gold F. Frederix,*,† K. Bonroy,† W. Laureyn,† G. Reekmans,† A. Campitelli,† W. Dehaen,‡ and G. Maes‡ IMEC, MCP-BIO, Kapeldreef 75, B-3001 Heverlee, Belgium, and KULeuven, Physical and Analytical Chemistry Department, Celestijnenlaan 200F, 3001 Heverlee, Belgium Received November 26, 2002. In Final Form: February 20, 2003 An affinity biosensor interface of a biosensor is the interface between the sample and the transducer surface and is therefore of the utmost importance for the general performance of a biosensor. For immunosensor applications the affinity biosensor interface consists of antibodies, which are preferably covalently attached to the transducer surface. In this paper the properties and the enhanced performance of an affinity biosensor interface based on mixed self-assembled monolayers (SAMs) on gold are discussed. Mixed SAMs consist of two different functionalities, which allow attachment of bioreceptor molecules and avoid nonspecific adsorption. In this work, mixed SAMs of thiols with carboxylic and hydroxyl or poly(ethylene glycol) groups are characterized with contact angle measurements, cyclic voltammetry, and grazing-angle Fourier transform infrared spectroscopy. It is found that the various mixed SAMs exhibit acceptable coverage and structural properties. Most importantly, surface plasmon resonance measurements clearly show the enhanced performance of these mixed SAMs with regard to sensitivity, stability, and selectivity compared to commercially available affinity biosensor interfaces. This superiority is experimentally demonstrated by evaluating the amount of immobilized antibodies, the recognition of antigens by the immobilized antibody, and the nonspecific adsorption of IgG molecules on the antibody-coated surfaces.
Introduction Biosensors provide a rapid and convenient alternative to conventional analytical methods for monitoring (bio)chemical substances in various fields such as medicine, environment, fermentation, and food processing.1-3 A biosensor basically consists of two parts, i.e., an affinity biosensor interface and a transducer. Since the affinity biosensor interface constitutes the interface with the sample, this component mainly determines the specificity, the reproducibility, and the stability of the whole sensor. In addition, nonspecific signals due to interferents constitute a major problem in diagnostic applications, where an analyte in a low concentration has to be detected in the presence of a much larger concentration of nonspecific molecules. The construction of a specific and stable affinity biosensor interface is therefore mandatory for real biosensor applications. The development of techniques for immobilization of the biomaterials plays a significant role in the biosensor research.4 The immobilization process not only ensures the intimate contact of the biological entities with the transducer but also aids in the stabilization of the biological system, enhancing its operational and storage stability. A number of methods have been applied for the * To whom correspondence may be addressed: phone, +32-16288013; fax, +32-16-281097; e-mail,
[email protected]. † IMEC, MCP-BIO. ‡ KULeuven. (1) Mulchandani, A.; Rogers, K. R. Enzyme and Microbial biosensors: Techniques and Protocols; Humana: Totowa, NY, 1998. (2) Ramsay, G. Commercial Biosensors: Applications to Clinical Bioprocess and Environmental Samples; John Wiley & Sons: London, 1998. (3) Nikolelis, D.; Krull, U.; Wang, J.; Mascini, M. Biosensors for Direct Monitoring of Environmental Polluants in Field; Kluwer Academic: London, 1998. (4) D’Souza, S. F. Appl. Biochem. Biotechnol. 2001, 96, 225.
immobilization of receptor biomolecules,5,6 e.g., adsorption,7 covalent attachment to silanes,8 embedding in polymers,9,10 and membranes.11 We have recently developed an affinity biosensor interface based on the use of mixed self-assembled monolayers (SAMs) of thiols on gold. Thiol compounds are known for their stable bond to gold and for their reproducible behavior.12 They may be used in applications such as electroanalytical chemistry,13,14 molecular electronics,15 corrosion research,16,17 biomaterial research,4,7,18 etc. In our approach, one of the thiols in the mixed SAM carries a functional group to attach the probe molecule, which is an antibody in immunosensor applications. The other thiol compound used for the mixed SAM construction is known to be adequate for limiting the nonspecific adsorption of undesired biological entities.19-23 The latter (5) Cass, T.; Ligler, F. S. Immobilized Biomolecules in Analysis: A practical Approach; Oxford University Press: New York, 1998. (6) Hermanson, G. T.; Mallia, A. K. Immobilized Affinity Ligand Techniques; Academic Press: London, 1992. (7) Castner, D. G.; Ratner, B. D. Surf. Sci. 2002, 500, 28. (8) Laureyn, W. Physicochemical study on the use of silanes for the realization of oxide-based biosensor interfaces. Ph.D. Thesis, KULeuven, Belgium, 2002. (9) Huang, N.-P.; Vo¨no¨s, J.; De Paul, S. M.; Textor, M.; Spencer, N. D. Langmuir 2002, 18, 220. (10) Cosnier, S. Biosens. Bioelectron. 1999, 14, 443. (11) Cooper, M. A. Nat. Rev. 2002, 1, 515. (12) Ulman, A. An Introduction to Ultrathin Organic Films; Academic Press: San Diego, CA, 1991. (13) Mandler, D.; Turyan, I. Electroanalysis 1996, 8 (3), 207. (14) Bard, A. J.; Abrun˜a, H. D.; Chidsey, C. E.; Faulkner, L. R.; Feldberg, S. W.; Itaya, K.; Majda, M.; Melroy, O.; Murray, R. W.; Porter, M. D.; Soriaga, M. P.; White, H. S. J. Phys. Chem. 1993, 97, 7147. (15) Matsui, H.; Porrata, P.; Douberly, G. E. NanoLett. 2001, 1 (9), 461. (16) Jennings, G. K.; Munro, J. C.; Laibinis, P. E. Adv. Mater. 1999, 11 (12), 1000. (17) Laibinis, P. E.; Whitesides, G. M. J. Am. Chem. Soc. 1992, 114, 9022. (18) Kasemo, B. Surf. Sci. 2002, 500, 656.
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thiol carries hydrophilic end groups such as hydroxyl or poly(ethylene glycol). The incorporation of both thiols in a mixed SAM enables the attachment of receptor molecules and induces specific interaction of the immobilized antibodies instead of a nonspecific adsorption of undesired biospecies on the surface. In this study, the antibodies are coupled by random, covalent coupling procedures in a direct or an indirect way via streptavidin and biotin.6 The antibody-antigen system applied to demonstrate the biosensor potential of these mixed SAMs is anti-human serum albumin (anti-HSA)-human serum albumin (HSA). It is well-known that the concentration of HSA in urine increases up to several hundred µg/mL in the case of kidney trouble. This implies that a urinary level in the order of several tens of µg/mL can be a marker for the diagnosis of diabetic nephrosis.24 In addition, this anti-HSA/HSA system is chosen as a cost-effective reference system for other antibody-antigen interactions. Experimental Section Materials and Methods. All materials and reagents were used as commercially received. Anti-human serum albumin (A0433), human serum albumin (A-1653), human IgG reagent (I2511), streptavidin (S-4762), 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) were obtained from Sigma. Biotinylated anti-human serum albumin was obtained from Biotrend Chemicalien Gmbh, while 11-mercapto-1-undecanol (11-MUOH) (>97%) and 16-mercapto1-hexadecanoic acid (16-MHA) (>90%) were purchased from Aldrich. Ultrapure ethanol was purchased from Riedel-DeHae¨n. Gold substrates mounted in chip (J1-chip) and the dextran-coated gold substrates (CM5-chip) were purchased from Biacore. NMR spectra were acquired on a Bruker Avance 300 MHz spectrometer. Chemical shifts (δ) are reported in parts per million referenced to internal residual solvent protons (1H) or the carbon signal of deuterated solvents (13C). Mass spectroscopy data were obtained with an HP MS apparatus 5989A (chemical ionization (CI), CH4) and with a MSSOTC KRATOS double focusing mass spectrometer in electron impact with perfluorokerosene as a reference compound under full magnetic scan mode. Preparation of Gold Substrates and Mixed SAMs. J1 chips were used as received and were immersed in the appropriate mixtures (v/v) of thiol solutions of 1 mM in ethanol immediately after opening. The reported mixtures are the percentages of thiols with carboxylic groups in the mixed thiol solution. After deposition for 3 h, the substrates were thoroughly rinsed with ethanol and dried under a stream of nitrogen. Initially, the deposition time was varied between 10 min and 24 h and a duration of 3 h was found to be optimal. The gold films for grazing angle Fourier transform infrared, contact angle, and cyclic voltammetry measurements were deposited by electron beam evaporation of 10 nm Ti and 100 nm Au on a polished 6′ Si wafer with 1.2 µm thermally grown SiO2. Surface Plasmon Resonance Spectroscopy. The surface plasmon resonance instrument was a Biacore 2000,25,26 provided with a J1-chip or CM5-chip. The immobilization degree of proteins (19) Chapman, R. G.; Ostuni, E.; Yan, L.; Whitesides, G. M. Langmuir 2000, 16, 6927. (20) Ostuni, E.; Yan, L.; Whitesides, G. M. Colloids Surf., B 1999, 15, 3. (21) Wang, R. L. C.; Kreuzer, H. J.; Grunze, M. J. Phys. Chem. B 1997, 101, 9767. (22) Lindblad, M.; Lestelius, M.; Johansson, A.; Tengvall, P.; Thomsen, P. Biomaterials 1997, 18 (15), 1059. (23) Silin, V.; Weetall, H.; Vanderah, D. J. J. Colloid Interface Sci. 1997, 185, 94. (24) Sakai, G.; Saiki, T.; Uda, T.; Miura, N.; Yamazoe, N. Sens. Actuators, B 1995, 24-25, 134. (25) Stenberg, E.; Persson, B.; Roos, H.; Urbaniczky, C. J. Colloid Interface Sci. 1991, 143 (2), 513. (26) Jo¨nsson, U.; Fa¨gerstam, L.; Ivarsson, B.; Johnsson, B.; Karlsson, R.; Lundh, K.; Lo¨fås, S.; Persson, B.; Roos, H.; Ro¨nnberg, I.; Sjo¨lander, S.; Stenberg, E.; Ståhlberg, R.; Urbaniczky, C.; O ¨ stlin, H.; Malmqvist, M. BioTechniques 1991, 11 (5), 620.
Frederix et al. in a Biacore surface plasmon resonance system is given in refractive units (RU). RU corresponds to a shift in resonance angle of approximately 0.1 millidegree.26 Immobilization of antibodies was accomplished via coupling to their primary amines. A continuous flow of Hepes buffered saline or HBS (10 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid, 150 mM NaCl, 3.4 mM ethylenediaminetetraacetate, and 0.005% Tween20 (pH 7.4)) at 5 µL/min was maintained during the immobilization period. All experiments were carried out at 25 °C. In detail, the carboxylic groups of the mixed SAMs were activated by injection of a solution containing different concentrations of NHS and EDC in deionized water. Next, 132 µL of the antibody solution (500 µg/mL in 10 mM acetate buffer pH 5.0) was injected, followed by injection of 50 µL of 1.0 M ethanolamine in order to block remaining NHS ester groups and by two 10 µL injections of 10 mM glycine (pH 2.2) in order to remove nonspecifically bound molecules from the surface. Antigens were diluted in HBS buffer to the concentrations applied and were then perfused over the antibody-immobilized surface at a flow rate of 20 µL/min. After 6.5 min of association, the sample solution was replaced by a HBS buffer flow for 7 min, allowing the complex to dissociate. Regeneration of the surfaces was performed by two pulses of 10 mM glycine (pH 2.2) between each analyte injection. The degree of binding was calculated by measuring the response signal at the end of the dissociation phase. The occurrence of nonspecific binding was measured by using 10 µg/mL IgG as the analyte. Grazing Angle Fourier Transform Infrared Spectroscopy. The mixed SAMs on gold were analyzed with grazing angle Fourier transform infrared spectroscopy (GA-FTIR) on a Mattson Galaxy series FTIR 7000, using a Spectra-Tech FT 85 accessory, over a wavenumber range of 5000-600 cm-1. The spectra are the result of a Fourier transform of 8960 interferometric scans at a resolution of 2 cm-1 on three different samples, which are subsequently averaged. The background sample was a SAM of HS-(CD2)17-CD3 on gold. This deuterated thiol, which is mandatory to perform this kind of FTIR measurement,19 was kindly supplied by the research group of Professor M. Grunze, Heidelberg, Germany. Contact Angle Measurements. Contact angle measurements (CA) were performed on 1 µL sessile drops of ultrapure water with an OCA 20 system from Dataphysics using SCA 20 software. Reported CA values are averaged over at least nine measurements, and the reported errors are 1 standard deviation. Cyclic Voltammetry. The cyclic voltammetry (CV) experiments were performed with a homemade electrochemical cell with a Pt counter electrode and an Ag/AgCl micro reference electrode from Microelectrodes, Inc. The setup uses a Gamry Instruments potentiostat with Framework software. All experiments were performed in a 6 mM K3Fe(CN)6 solution with 1 M KCl as the background electrolyte. Synthesis of 2-(2-(2-(6-Mercaptohexyloxy)ethoxy)ethoxy)ethanol. 2-(2-(2-(6-Bromohexyloxy)ethoxy)ethoxy)ethanol was synthesized using the procedure described by Flink.27 Fifteen grams of tri(ethylene glycol) (Aldrich, 99%) and 1.45 g of NaH (Acros, 60% in mineral oil) were dissolved in dry DMF and stirred for 50 min. The resulting solution was treated with 100 g of dibromohexane (Aldrich, 96%) and subsequently stirred for 36 h. This mixture was quenched with methanol. After evaporation of the solvent, the resulting oil was dissolved in 250 mL of CH2Cl2, washed four times with water, and dried over MgSO4. Subsequently, the solvent was evaporated and the residue was purified by column chromatography (SiO2, 2:1 EtOAc/hexane) yielding 2-(2-(2-(6-bromohexyloxy)ethoxy)ethoxy)ethanol (9.624 g, 85%) as verified with 1H NMR and mass spectroscopy. 2-(2-(2-(6-Mercaptohexyloxy)ethoxy)ethoxy)ethanol was synthesized from the previous compound using the method described by Bader.28 2-(2-(2-(6-Bromohexyloxy)ethoxy)ethoxy)ethanol and 13.8 g of thioureum (Aldrich, 99%) were dissolved in ethanol and refluxed for 22 h under a N2 atmosphere. The resulting solution was treated with 2 g of NaOH in a few mL of water and refluxed (27) Flink S. Sensing Monolayers on Gold and Glass; Twente: Netherlands, 2000. (28) Bader, M. M. Phosphorus, Sulfur Silicon Relat. Elem. 1996, 116, 77.
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Figure 1. Schematic representation of the mixed SAMs consisting of 16-mercapto-1-hexadecanoic acid and 11-mercapto-1-undecanol, i.e., SAM1 (a), and of 16-mercapto-1-hexadecanoic acid and 2-(2-(2-(6-mercaptohexyloxy)ethoxy)ethoxy)ethanol, i.e., SAM2 (b).
Figure 2. Cyclic voltammograms for different ratios of 16-mercapto-1-hexadecanoic acid and 11-mercapto-1-undecanol in the mixed SAM1 on the gold electrodes. Measurements were performed in 6 mM K3Fe(CN)6 in 1 M KCl with a scan rate of 0.1 V s-1. for an additional 20 h under a N2 atmosphere. The mixture was then treated with HCl in ice-cold water, dissolved in 100 mL of CH2Cl2, and washed with water. The solvent was evaporated, and the oil-like residue was purified by column chromatography (SiO2, 15:1 CH2Cl2/MeOH) giving rise to 2-(2-(2-(6-mercaptohexyloxy)ethoxy)ethoxy)ethanol or 6-PEO (4.16 g, 52%). 1H NMR (300 MHz, CDCl3): δ ) 1.38 (m, 4H), 1.60 (m, 4H), 2.52 (q, 2H), 3.09 (br, 1H), 3.45 (t, 2H), 3.65 (m, 12H). 13C NMR (75 MHz, CDCl3): δ ) 24.3, 25.3, 27.9, 29.2, 33.7, 61.4, 69.8, 70.1, 70.3, 70.3, 71.1, 72.3. MS (CI): 267.4 (MH+), 151, 117. MS (EI) (M + H) ) 267.16317 with an elemental composition of C12H27O4S and an error of -0.2 mmu.
Results and Discussion Surface Characterization of the Mixed SAMs. A schematic presentation of the two mixed monolayers studied, 16-MHA and 11-MUOH (SAM1) or 16-MHA and 6-PEO (SAM2), is shown in Figure 1. The structural characterization of the monolayers was performed using CV, CA, and GA-FTIR. The CV results illustrated in Figure 2 demonstrate that mixed SAMs of 16-MHA and 11-MUOH form densely packed monolayers after 3 h of deposition time. In addition it is observed that the oxidation/reduction peak separation increases with increasing concentrations of 16-MHA in the mixed SAM1. The higher alkyl chain length of the latter thiol compound probably allows the mixed SAM to become more densely packed, i.e., the redox couple K3Fe(CN)6 is further away from the working electrode, which
gives rise to a larger peak separation (Figure 2).29 Similar voltammograms have been observed for SAM2. CA measurements give additional information on the physical properties of the mixed SAM surfaces. The water contact angle of mixed SAM1 with 5% of 16-MHA is very small, i.e.,
